U.S. patent number 7,842,465 [Application Number 11/333,731] was granted by the patent office on 2010-11-30 for immunocytostaining methods for enhanced dye ratio discrimination in rare event detection.
This patent grant is currently assigned to Palo Alto Research Center Incorporated. Invention is credited to Richard H. Bruce, Douglas N. Curry, Huangpin Ben Hsieh, Robert T. Krivacic, Nicole H. Lazarus.
United States Patent |
7,842,465 |
Hsieh , et al. |
November 30, 2010 |
Immunocytostaining methods for enhanced dye ratio discrimination in
rare event detection
Abstract
A method is provided for preparing a sample containing potential
cells of interest and of using a laser of a laser based system for
novel excitation and emission collection, and data usage including
use of obtained data for direct and ratio based measurements. The
prepared sample is configured to emit signals having spectral
characteristics sufficient to permit filtering to differentiate and
eliminate most false positives from true positives among acquired
imaging events, in an imaging system employing a laser spot having
a range of diameters from 1 to 20 .mu.m or greater and which
excites the fluorescence in a conventional or novel manner. These
filtered events may be subsequently imaged and confirmed with
another higher resolution device such as a fluorescent microscope
in a short amount of time.
Inventors: |
Hsieh; Huangpin Ben (Mountain
View, CA), Lazarus; Nicole H. (Sonoma, CA), Krivacic;
Robert T. (San Jose, CA), Curry; Douglas N. (Palo Alto,
CA), Bruce; Richard H. (Los Altos, CA) |
Assignee: |
Palo Alto Research Center
Incorporated (Palo Alto, CA)
|
Family
ID: |
37989191 |
Appl.
No.: |
11/333,731 |
Filed: |
January 17, 2006 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20070166770 A1 |
Jul 19, 2007 |
|
Current U.S.
Class: |
435/7.1; 436/532;
424/178.1; 424/130.1 |
Current CPC
Class: |
G01N
21/6428 (20130101); G01N 33/56966 (20130101); G01N
1/30 (20130101); G01N 15/1468 (20130101); G01N
21/6456 (20130101); G01N 21/6458 (20130101); G01N
2201/0833 (20130101); G01N 2021/6482 (20130101); G01N
2021/6441 (20130101); G01N 2021/6419 (20130101); G01N
2021/6421 (20130101); G01N 2021/6484 (20130101) |
Current International
Class: |
G01N
33/53 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Krivacic, Robert T., et al., "A rare-cell detector for cancer",
Proceedings of the National Academy of Sciences of the U.S. of
America, Jul. 20, 2004, vol. 101, No. 29, pp. 10501-10504. cited by
other .
Curry, D. N., et al., "High-speed detection of occult tumor cells
in peripheral blood", Proceeding sof the 26.sup.th Annual
International Conference of the IEEE EMBS, San Francisco, CA, Sep.
1-5, 2004, pp. 1267-1270. cited by other .
Hsieh, H. Ben, et al., "High speed detection of circulating tumor
cells", Biosensors & Bioelectronics, Elsevier Science
Publishers, Barking, GB, vol. 21, No. 10, Apr. 15, 2006, pp.
1893-1899. cited by other .
European Search Report, Application No. 07100507.8-2204; Dated Jun.
12, 2007, Munich, Germany. cited by other.
|
Primary Examiner: Yang; N C
Attorney, Agent or Firm: Fay Sharpe LLP
Claims
The invention claimed is:
1. A method of preparing a sample containing potential cells of
interest, for use in a laser based scanning system designed to
detect imaging events which contain the potential cells of
interest, the method comprising: placing a first material
containing the potential cells of interest on a slide; adding at
least a first type of antibody configured to target the potential
cells of interest and allowing binding of the first type of
antibody to proceed to completion; adding a second type of antibody
configured to target the first type of antibody; associating at
least a first marker or tag, in a first amount, with at least one
of the first type of antibody and the second type of antibody; and
associating a second marker or tag, in a second amount different
than the first amount, with at least one of the first type of
antibody and the second type of antibody, wherein when acted upon
by the laser based scanning system, the prepared sample is
configured to emit signals having a spectral resolution sufficient
to permit filtering to differentiate and eliminate most false
positives from true positive imaging event, the filtered imaging
events are then confirmed by a higher resolution detection method
to determine whether they are truly the cells of interest.
2. The method of claim 1, wherein there are a plurality of the
second type of antibody, the first marker or tag associated with a
first subset of the plurality, and the second marker or tag
associated with a second subset of the plurality.
3. The method of claim 2, wherein the first marker or tag
associated with the first subset of the second type of antibody,
and the second marker or tag associated with the second subset of
the second type of antibody compete for association with the first
type of antibody.
4. The method of claim 1, wherein the first marker or tag is
associated with the first type of antibody and the second type of
marker or tag is associated with the second type of antibody.
5. The method of claim 4, wherein the second type antibody with the
associated second marker or tag is targeted to the first type
antibody, in a procedure which is a sequential non-competitive
manner.
6. The method of claim 1, wherein the first and second markers or
tags are each provided in a concentration which is diluted in a
range of six to seven times less than an industry understood
concentration level for cell detection.
7. The method of claim 1, wherein at least one of the first marker
and second marker or tag is Alexa 555 provided at a concentration
of approximately 150.times. dilution.
8. The method of claim 1, wherein at least one of the first marker
or tag or the second marker or tag are selected to exhibit a
substantially non-optimal excitation for the wavelength of a
selected laser of the laser based scanning system to increase
spectral differences between the emitted signals.
9. A method of preparing a sample containing potential cells of
interest, for use in a laser scanning system designed to detect
imaging events which contain the potential cells of interest, the
method comprising: placing a first material containing the
potential cells of interest, on a slide; associating a first marker
or tag to a first type of antibody; associating the first type of
antibody to a potential cell of interest; and associating a second
marker or tag to a second type of antibody, the second type of
antibody configured to target the first type of antibody, wherein
the first marker or tag and second marker or tag are associated
with the respective first type of antibody and second type of
antibody in a sequential non-competitive manner and wherein when
acted upon by the laser based scanning system, the prepared sample
is configured to emit signals having a spectral resolution
sufficient to permit filtering to differentiate and eliminate most
false positives from true positive imaging event, the filtered
imaging events are then confirmed by a higher resolution detection
method to determine whether they are truly the cells of
interest.
10. The method of claim 9, wherein there is a larger number of the
second type of antibody with the associated second marker or tag,
than the first type of antibody with the associated first marker or
tag to increase a spectral difference between signals emitted from
the first marker or tag and the second marker or tag, when the
sample is scanned by a laser.
11. A method of preparing a sample containing potential cells of
interest, for use in a laser scanning system designed to detect
imaging events which contain the potential cells of interest, the
method comprising: placing a first material containing the
potential cells of interest on a slide; adding a first type of
antibody configured to target the potential cells of interest;
adding a second type of antibody configured to target the first
type of antibody; associating a first marker or tag with the first
type of antibody; and associating a second marker or tag with the
second type of antibody, wherein the first and second markers or
tags are provided at a predetermined concentration ratio to create
an asymmetric marker or tag arrangement that provides a desired
spectral resolution comprising an emission intensity ratio of the
first and second markers or tags; exciting the first and second
markers or tags by the laser based scanning system, causing the
markers or tags to emit a first signal from the first marker or
tag, and a second signal from the second marker or tag, the first
signal and the second signal having spectral differences from each
other of a spectral resolution sufficient to permit filtering to
differentiate and eliminate false positives from a true positive
imaging event and filtering imaging events that fall below a
predetermined level of emission intensity ratio; and using a higher
resolution detection method to determine whether they are truly
cells of interest.
12. The method of claim 11 wherein the spectral resolution
sufficient to differentiate false positives is achieved using only
the first type of antibody and the second type of antibody, the
first type of antibody and the second type of antibody being
different from each other.
13. The method of claim 11, wherein the second type of antibody
targets the first type of antibody and does not target the first
marker or tag.
14. The method of claim 11, wherein an amount of the first marker
or tag and an amount of the second marker are not equal and are
selected to obtain the spectral differences sufficient to permit
the filtering to differentiate false positive.
15. The method of claim 11 wherein the placing step includes
binding the first type of antibody to the cells of interest.
16. The method of claim 11, wherein the second type of antibody
with the associated second marker or tag is targeted to the first
type of antibody, in a procedure which is a sequential
non-competitive manner.
17. The method of claim 11, wherein at least one of the first
marker or tag or the second marker or tag are selected to exhibit
less than optimal excitation for a wavelength of a selected laser
of the laser based scanning system to increase spectral differences
between the emitted first signal and the emitted second signal.
Description
BACKGROUND
The present application relates to laser based detection systems,
and finds particular application in conjunction with low and
high-density cell detection and discrimination in blood smears,
biological assays, and the like, and will be described with
particular reference thereto. However, it is to be appreciated the
present concepts will also find application in detection and
discrimination of other types of low- or high-density features on
various substantially planar surfaces and samples.
Laser based detection systems are widely used in many industries,
including printing, bio/life and medical sciences, and are
implemented in biochip readers, and laser scanning cytometers,
among other detection systems.
In order to achieve high resolution in one category of such
devices, laser light is guided through objectives similar to those
for microscopes. These objectives utilize multiple lens elements to
achieve high magnification and often near- or sub-micron
resolution. Since both excitation and emission light are guided
through these objectives, the heavy weight of the objectives and
their small aperture limits the speed at which the laser light can
be moved and thus limits the speed of scanning of a sample.
Fiber Array Scanning Technology (FAST) developed by Palo Alto
Research Center (PARC) of Palo Alto, Calif. does not utilize a
microscope-type objective. Instead FAST employs a rapid spinning
galvanometer or mirror for directing laser light and a
large-aperture fiber bundle to collect light emission over a
relatively large area. The FAST scanning speed is very high,
however, its spatial resolution is currently at a laser spot of
approximately 8 .mu.m. Concepts of FAST based systems is described,
for example, in published U.S. Patent Applications 2004/0071330
("Imaging Apparatus and Method Employing a Large Linear Aperture")
and 2004/0071332 ("Apparatus and Method for Detecting and Locating
Rare Calls") to Bruce et al. (each hereby incorporated in their
entirety by reference).
The implication of the relatively low spatial resolution (i.e.,
several .mu.m) is the inability of FAST to detect detailed cellular
structures or staining characteristics. Therefore, detected matter
other than true positive cells, which often have similar intensity
and size of true positives, can therefore register as a potential
hit. Even after some rudimentary filtering, such as size/intensity
scrutiny, an undesirably large number of potential hits still need
to be investigated, using time-consuming microscopy investigation.
The occurrences of these false positive hits strongly depend on
sample preparation methodology where present techniques do not
result in sufficient spatial resolution of a detected image event
to differentiate between a false positive and a true positive when
FAST type system is used.
BRIEF DESCRIPTION
A method is provided for preparing a sample containing potential
cells of interest and of using a laser of a laser based system for
novel excitation and emission collection, and data usage including
use of obtained data for direct and ratio based measurements. The
prepared sample is configured to emit signals having spectral
characteristics sufficient to permit filtering to differentiate and
eliminate most false positives from true positives among acquired
imaging events, in an imaging system employing a laser spot having
a range of diameters from 1 to 20 .mu.m or greater and which
excites the fluorescence in a conventional or novel manner. These
filtered events may be subsequently imaged and confirmed with
another higher resolution device such as a fluorescent microscope
in a short amount of time.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a perspective view of an imaging apparatus formed in
accordance with an embodiment of the present application.
FIG. 2 illustrates a diagram depicting a labeling scheme employing
two secondary antibodies in a competitive acquisition method;
FIG. 3 depicts excitation and emission spectra of Alexa dyes;
FIG. 4 shows a labeling scheme employing a sequential application
of conjugated primary and a conjugated secondary method;
FIG. 5 depicts a labeling scheme which employs Tyramid Signal
Application for application of a signal;
FIG. 6 depicts a further procedure for improving spectral
resolution employing a single dye configuration; and
FIG. 7 illustrates the output of the use of a dual dye
amplification procedure wherein a ratio of output signals is used
for improving the spectral resolution of a sample.
DETAILED DESCRIPTION
With reference to FIG. 1, one embodiment of an imager 10, employing
the Fiber Array Scanning Technology (FAST), is depicted. Imager 10
examines a sample 12 with biological smear 14 disposed on at least
a portion of a surface of a slide 16.
As is known in the art for cell studies, sample 12 is prepared by
drawing a sample of a biological fluid such as, but not limited to,
blood or parts of blood from a subject. In a preferred embodiment,
the sample is a monolayer of cells adhered to a slide. In
particular, blood cells with red cells removed. The fluid sample is
treated with a fluorescent material, such as but not limited to a
biological marker conjugated to dye fluorphore that selectively
bonds to different kinds of biological molecules, which may be on
the surface or inside the cell, such as proteins, nucleic acids or
other molecules. Suitable markers are known in the art for marking
a number of different cell types of clinical interest, including
selected cancer cell types, fetal cells, or other appropriate cells
to be considered. Work is also being undertaken to develop marking
materials for numerous cell types from other organs such as brain
cells, liver cells, as well as bacteria cells or viruses, among
others. The marking material preferably emits a characteristic
luminescing output, such as a fluorescence or phosphorescence
light, responsive to a selected excitation irradiation, such as
irradiation by a selected wavelength or spectrum of light, x-ray
irradiation, electron-beam irradiation, or the like. The
characteristic luminescence typically has a characteristic
wavelength or spectral range of wavelengths. While organic dyes
(i.e. fluorphores) are the predominant tagging process, other
techniques exist including the use of other markers known as
quantum dots and nano-particle probes. Systems using these as well
as other materials and techniques may beneficially employ the
concepts of the present application.
The smear size will depend on implementation, however, as an
example, in a biological fluid, in one situation for a rare cell
concentration of about one rare cell of interest per one million
cells, the smear 14 might contain one million to 50 million or more
cells and occupy an area of about 10 cm.sup.2 to 100 cm.sup.2 or
greater. Of course, larger or smaller smears can be prepared which
are suitable for the anticipated concentration of cells in the
sample and the desired minimum measurable cell concentration.
Sample 12 is mounted on an imager translation stage 18 (shown in a
partial view) which includes a linearly translatable track 20 that
supports sample 12. A motor 22 connects with track 20 via gearing
24 to translate track 20 and the supported sample 12 along a
y-direction (indicated by arrows 28) in an x-direction (indicated
by arrows 28).
A light pipe 30, such as a fiber optic bundle, includes a first end
32 that is proximate to the sample 12, and a second end 34 that is
distal from the sample 12. The first end 32 includes a plurality of
first fiber ends arranged substantially parallel to one another in
an arrangement that defines a generally linear or high-aspect-ratio
rectangular input aperture 36 with a long dimension aligned with
the x-direction.
The optical fiber bundle 30 "morphs" or changes cross-sectional
dimensions and shape between the first end 32 to the second end 34
such that the second end 34 includes a plurality of second fiber
ends that define a compact, generally circular output aperture
38.
A scanning radiation (light) source 40 in a suitable embodiment
includes a laser 42 that produces excitation light (radiation beam)
44 at a wavelength or wavelength range selected to excite the
marking material used in marking the biological smear 14. The
excitation light 44 is angularly scanned by a galvanometer 46 that
has a reflective surface that rotates (indicated by curved arrows
48) responsive to an electrical input. An optional focusing lens 50
focuses the angularly scanned excitation light 64 onto the sample
12, and more particularly onto the biological smear 14. The angular
scanning produced by the galvanometer 46 translates into a linear
sweeping or fast scanning (indicated by arrows 52) of the
excitation light, preferably in the form of a spot, which presently
is approximately 8 .mu.m or greater in diameter on the biological
smear 14 along a linear trajectory 54 arranged below the input
aperture 36 and parallel to the long dimension of the input
aperture 36.
An electronic control unit 56 communicates with the galvanometer 46
and the translation stage 18 to coordinate the linear sweeping or
scanning 52 of the radiation beam 44 along the trajectory 54 and
the linear translation 26 of the sample 12 to effectuate a
rastering of the radiation beam 44 across a selected area of the
sample which is bounded in the x-direction by the smaller of a span
of the trajectory 54 and the long dimension of the input aperture
32. Preferably, the span of the trajectory 54 substantially
comports with the long dimension of the input aperture 32.
The scanning radiation source 40 and the input aperture 36 are
arranged in fixed relative position, the galvanometer 46 provides a
linear sweeping of the excitation beam 44 along the x-direction,
and the sample 12 is moved by the translation stage 18 linearly
along a y-direction to effectuate a two dimensional rastering.
A suitable signal detector 58 is arranged to detect the collected
characteristic luminescence emanating from the output aperture 38.
A first lens 60 substantially collimates the light, such as but not
limited to a laser light. Blocking filter 62 is optionally provided
to remove scattered laser light from the collected light.
A second lens 64 may be provided to focus the collimated collected
light onto a photodetector arrangement 66 Combining the compact
output aperture 38 with focusing optics 60, 64, photodetector 66,
which may be a single photodetector, provides signal detection for
the spatially distributed linear input aperture 36. Because of the
typically low collected characteristic luminescence intensities
produced by treated cells, the photodetector 98 is preferably a
photomultiplier tube.
Electronic control unit 56 communicates with the galvanometer 46
and the translation stage 18 to raster the radiation beam 44 across
the sample. Characteristic luminescence produced by interaction of
the radiation beam 44 with treated cells in the biological smear 14
is collected by the input aperture 36, channeled to the output
aperture 38 by the optical fiber bundle 30, and detected by the
signal detector 58. The electronic control unit 56 receives the
detected signal from the photodetector 66, and correlates the
detected signal with positional coordinates of the radiation beam
44 on the sample 12.
The electronic control unit 56 suitably formats the detected signal
and spatial coordinates information and stores the information in
an internal memory, writes the information to a non-volatile
storage medium such as a magnetic or optical disk, formats and
displays an image representation including an array of picture
elements with coordinates mapped to the spatial coordinates
information and an intensity or color mapped to the detected signal
intensity on a display 68, or the like.
As previously discussed, during the scanning operations,
interaction of the spot generated by the laser beam with tagged
cells in a sample will cause those tags (or markers) to emit a
luminescence, such as a fluorescent light. Commonly, these tags are
clustered within the cells and generate high-intensity pixels when
they are excited and reemit upon scanning by the radiation spot.
For the following discussion, the detected unknown cluster of tags
is described as an "image event" to which further investigation is
warranted. The size of the radiation spot defines the resolution of
the imaging device.
When working with such small structures, noise--such as dirt or
dust particles, or miscellaneous cells--may be found on the sample
12, and will have an effect on the acquired image information.
Specifically, the imager 10 may accumulate image data irrelevant to
the identification of rare cells. At times this noise may be
detected as "false positives." It is desirable to eliminate this
noise during image acquisition and processing. Therefore, filtering
procedures are implemented via electronic control unit 80 and/or
other elements of system 10 to eliminate information not related to
rare cells. The filtering techniques may use various
characteristics of an image event to perform the filtering
operations, including the number of pixels, intensity, phase and
shape of the image event under consideration.
In one embodiment, an image event may be classified as a non-rare
cell (false positive) or a rare cell (true positive) image event by
counting the number of pixels of the image event under
investigation. Knowing approximate sizes of rare cell tag clusters
under investigation, a range can be set to filter out those image
events having either a number of pixels less than or greater than
the prescribed range. For instance, if the range of rare cells
would be known to correlate to a number of pixels in a range of 1
to 12, then image events having a pixel range greater than 12,
would be eliminated in a filtering operation.
In another filtering embodiment, the shape of an image event is
used to filter non-relevant information. Specifically, in many
instances an image event correlating to a rare cell or cluster of
rare cells would have a known shape corresponding to the rare cells
being imaged, and blurred by the impulse response of the radiation
spot. If the detected shape is other than expected for the
pertinent rare cell and/or clusters of rare cells, this would
indicate the detected image event is noise such as a dust or dirt
particle or other irrelevant signal from the sample. To assist in
the filtering in this arrangement, known pattern matching software
may be implemented in imaging system 10. In this filtering
operation, it would be expected not to see an image event that had
a finer structure than the spots own resolution size. Particularly,
the image event would not be smaller than the spot size, although
the structure itself may be smaller.
Still a further filtering process which may be used to identify
rare cell image events from non-rare cell image events is by
tracking the intensity of the image event under investigation. For
example, it would be expected that a higher intensity would be
detected for rare cell image events that are in phase with the
pixel acquisition phase, and would also provide fewer pixels. Out
of phase image events would have their energy shared with several
neighboring pixels, thereby providing a smaller intensity per
pixel, but more pixels. In addition, in some non-specific binding
of tags on cells, i.e., cells not related to the rare cells, may
produce image events but these would have a lower intensity than
the expected intensity from rare cell binding clusters.
The foregoing describes filtering techniques which may be used to
screen for rare cell events (true positives) from the image events
detected by the imaging system.
However, while some false positives can be detected and eliminated
from consideration by employing the above-described filtering
concepts, many of the false positives will not be eliminated since
the filtering criteria used cannot be set at too stringent of a
level. For example, when the filter employs size/intensity
criteria, the specific parameters for the size/intensity values
cannot be set at a level where all false positives are eliminated,
due to the potential adverse effect on true positives.
Particularly, in one situation, staining patterns of occult cancer
cells are irregular and are imprecise or unknown and, therefore,
would be missed if strict (i.e., narrow) size/intensity criteria
were set. Thus, existing filtering techniques are not capable of
lowering the amount of false positives which need to be
investigated to a manageable amount. One particular reason for this
inability is due to insufficient spatial resolution with a
FAST-type laser based imaging technique mentioned above.
A manner to address the issue of insufficient spatial resolution in
a large view/fast scanning system, such as FAST, is to improve
sample preparations in a manner whereby during the detection
procedure spectrally distinguishable characteristics of the sample
that are unique to true positive cells are emitted.
Thus, in addition to and/or in combination with the above discussed
filtering techniques, false positives may be minimized in a FAST
based type system by improving the spectral characteristics of the
prepared samples, to permit easier discrimination between image
events.
In conventional fluorescent microscopy (i.e., non-FAST type
imagers), broadband excitation sources (e.g., high pressure mercury
arc lamp, xenon lamp) are used with bandpass or longpass filters
optimized for exciting specific fluorophores (or dyes), in
particular in multiple-color microscopy application. The fluorphore
can be pre-conjugated to primary antibody so that when put together
they bind to the cellular target (antigen), the whole assembly
fluoresces when properly illuminated. Alternatively, a
fluorphore-conjugated secondary antibody can be used; this
secondary is immunized with the serum from the host in which the
primary is raised, so it will specifically target the primary
antibody. Using the secondary antibody has the advantage of a
brighter signal due to signal amplification because multiple copies
of the secondary antibody can bind to a single primary antibody,
thereby providing for increased ease of discrimination of image
events.
In a laser-based scanning system, the laser spot size is usually in
the range of a few to tens of microns. In PARC's FAST system, the
spot size is approximately 8 .mu.m or greater in diameter, which is
close to the size of a human blood cell (.about.10 .mu.m) and a
typical occult circulating cancer cell. It therefore is not able to
provide sub-micron resolution needed to view cellular structural
characteristics. Although using raster scanning with a galvanometer
in a FAST imager achieves high speed, it is at the expense of this
lower resolution--a resolution not sufficient to distinguish
whether a particular image event represents a false positive which
may be an auto-fluorescent artifact, dye aggregate or a true
positive, i.e. a genuinely labeled cellular target.
Thus, the analysis of the size and intensity for a single band of
fluorescence and/or use of the discussed filtering techniques alone
may not be sufficient for effective false positive elimination, as
many other matters could give fluorescence pattern similar to those
of true positives, i.e. their Stoke's Shift is similar or identical
to that of the fluorphores attached to true positives. Where a
Stokes Shift is known to be the difference in wavelength between
absorbed and emitted quanta. The emitted wavelength is longer or
equal to the incident wavelength due to energy conservation, the
difference being absorbed as heat in the atomic lattice of the
material.
Other matters which could affect fluorescence patterns include auto
fluorescence from the blood sample and tiny dust particles or dye
aggregates. For example, it is assumed an anti-cytokeratine primary
antibody (mouse anti-human) is used at 1:100 dilution to target
potential epithelial (likely cancerous) cells circulating in the
blood stream, and a secondary antibody such as Alexa 488-conjugated
goat anti-mouse is used at 1:1000 dilution to identify the targeted
cells. In a typical sample prepared and scanned by a FAST type
imager, thousands or more potential hits or image events may be
registered at the regular emission band of Alexa 488, i.e. 525
nm+/-20 nm. However, only a few or perhaps none may be true
positives. The differences in intensity and/or spot sizes between
true positives and false positives are simply not sufficiently
distinct to be distinguishable. This inability to distinguish the
false positives results in numerous hours of microscopy
investigation to image potentially thousands of these false
positives. Therefore, in order to better discriminate true
positives from false positives novel strategies in the way the
fluorescent antibody conjugates are applied in a sample are
implemented, thereby generating additional information to improve
the filtering applied to screen out the false positives.
Particularly, improved spectral characteristics of a sample may be
achieved by using unconventional excitation and emission methods to
eliminate background fluorescence and by employing multiple markers
(e.g., dyes, etc.) in various ways that target the same cells to
create emission ratio signatures that uniquely identify true
positives. An unconventional excitation and emission, in one
instance, includes employing a laser and a marker or tag together
which does not result in an optimal fluorescing of the marker or
tag. In the following discussion, the examples refer to the marker
as a fluorphore dye, however it is to be understood other
appropriate markers are also appropriate for use in connection with
the present concepts.
It is to be appreciated that while the foregoing has discussed the
FAST system, the following strategies may be applicable to other
luminescent (e.g., fluorescent, etc.) systems that use a single
laser for excitation. Furthermore, the described strategies and
concepts may also be employed in systems employing two or more
lasers to increase the specificity by using the first laser while
the other laser(s) are freed-up for exciting other tags for
additional information.
In a first implementation illustrated in FIG. 2, instead of using
just one fluorophore conjugated (i.e., associated) to the secondary
antibody, two are used. Particularly, this procedure illustrates a
primary antibody (1.sup.st Ab) 70 which is bound to a cellular
target 72 of interest, by a known technique. The cellular target
being part of material having been placed on a slide with a sample
such as in FIG. 1. Then multiple secondary antibodies 74 (of a same
type) having two different versions of fluorphores 76(*), 78(+)
selectively conjugated thereto. Both the conjugated fluorophores
76, 78 are designed to target the same primary antibody 70.
When used at different concentrations, their emission intensity
reflects the concentration used. For example, Alexa 488 goat
anti-mouse and Alexa 555 goat anti-mouse may be used to target and
compete for primary mouse anti-human cytokeratin. However, as shown
in Table 1 below, which lists quantum properties of selected Alexa
dyes, and as illustrated in FIG. 3, which depicts excitation and
emission curves of the Alexa dyes 488 and 555, when a 488 nm laser
is used, Alexa 488 is excited at much higher efficiency (75.1%
according to data from Molecular Probe/invitrogen Corporation of
Carlsbad, Calif.) than Alexa 555 (14.2%). In FIG. 3 the excitation
and emission spectra of Alexa dyes (EX.sub.--488 (80), EX.sub.--555
(82), EM.sub.--488 (84), and EM.sub.--555 (86)) are plotted by
absorbance or emission efficiency (Percent Efficiency) versus
wavelength (Wavelength (nm)).
TABLE-US-00001 TABLE 1 Alexa488 Alexa555 Ex Peak (nm) 499.0 553.0
Em Peak (nm) 520.0 568.0 Exit. Coef. 7.1E+04 1.5E+05 488 nm Ex.
Efcy (%) 75.1 14.2
With continuing attention to the first procedure of the present
application, to improve the spectral resolution of the sample, a
greater amount of Alexa 555 (e.g., 16) antibody (specifically, goat
anti-mouse antibody conjugated to Alexa 555) is used than Alexa 488
antibody (e.g., 14). More particularly, Alexa 555 (16) is provided
at a concentration ratio of 100:1 as compared to the Alexa 488 (18)
antibody. This makes it possible to create an Alexa 555 emission
intensity (and/or the amount of emitted photons that can be
collected) that's above that of Alexa 488 when excited by the 488
nm laser (such as laser 42 of FIG. 1). Of course other
concentration ratios may be applied, in order to create the
asymmetric marker arrangement, which provides the desirable
spectral resolution.
Further, blood auto fluorescence, for example, would typically have
an emission peak around that of Alexa 488 (.about.525 nm) when
excited by a 488 nm laser, and very little or none at that of Alexa
555 (.about.580 nm). Testing of various cancer cell lines when
excited by one 488 nm laser have shown that virtually all of
determined true positives have emission intensity ratios (580 nm
vs. 525 nm; in the form of average intensity per pixel) greater
than 1.0, with the majority of the true positives at 2 or above,
when the Alexa 555 dye is used at 100:1 over Alexa 488 dye. The
majority of false positives were determined to have a ratio below
1.0. Therefore, "image events" with emission ratios of 1.0 or below
may be disregarded and not imaged by the use of a higher resolution
device, such as a fluorescent microscope. Some false positives
stemming from random aggregates of secondary antibodies or
non-specifically bound primary might also have a >1.0 emission
ratio and thus will not be filtered out using the above procedure.
While the tested cancer cell lines have found the above ratios to
be relevant, it is understood other cell lines or patient samples
may have different ratios relevant to false positives and true
positives. Therefore, for these cell samples, the ratios obtained
by testing of these other cell samples would of course be used for
filtering.
Thus, by using the above-described "asymmetric" secondary
antibody-dye concentrations and selecting an appropriate emission
ratio cutoff as a filtering parameter, the amount of false
positives are significantly reduced.
Although the above asymmetric concept provides improved detection
and elimination of false positives, there are some issues
associated with this procedure. Particularly, the low excitation
efficiency of Alexa 555 by a 488 nm laser, combined with a bleeding
of Alexa 488 emission to Alexa 555 emission area (.about.580 nm,
see the tail of Alexa 488 emission, e.g., EM.sub.--488 (84) in FIG.
3) requires the use of a substantially high enough amount of the
Alexa 555. Furthermore, in order to avoid a weak signal and
therefore non-detection, Alexa 488 concentration cannot be too much
lower than a regular or commonly used 1000.times. dilution. For
example, Alexa 488 may be used at a 3000.times. dilution while
Alexa 555 at 30.times. dilution to substantially ensure a high
probability that all true positives have an emission ratio >1.0.
However, a 30.times. dilution of a typical secondary antibody stock
(e.g. 2 mg per mL) is considered "very concentrated" which also
increases the likelihood of more false positives due to
non-specific staining.
A second issue in the asymmetric implementation is that the
competitive nature of two secondary antibodies 76, 78 binding to
single primary antibody 70 means the results of such bonding can
sometimes be less than consistent across different experiments,
especially in cells with a low antigen expression level that can be
easily saturated.
Turning to FIG. 4, depicted is a second approach used to improve
the spectral distinctiveness of a sample. In this second approach,
instead of using two fluorophore-conjugated secondary antibodies,
one (or a first) fluorphore 90(+) is conjugated to a primary
antibody 92, which is bound to a cell of interest 94. Another (or
second) fluorophore 96(*) is conjugated to a secondary type
antibody 98, selected and prepared to target the
fluorphore-conjugated primary (or 1.sup.st type) antibody 92. As
depicted in FIG. 4, this binding is sequential and non-competitive
and can be allowed to go to completion.
A specific example of the above employs Alexa 488 mouse anti-human
cytokeratin (as the primary antibody) 92 and Alexa 555 goat
anti-mouse (as the secondary antibody type) 98. In this scheme, the
primary conjugated fluorphore (Alexa 488) 90 is the weaker channel
while the signal of the secondary antibody conjugated fluorphore
(Alexa 555) 96 is amplified.
Turning to a third approach, instead of using a high secondary
antibody concentration for the weakly excited channel (e.g., Alexa
555) as discussed in connection with FIG. 2, the concept shown in
FIG. 5 depicts a procedure which uses an enzyme based signal
amplification method, such as the Tyramide Signal Amplification
(TSA) to amplify the secondary antibody (e.g., Alex 555).
Tyramide Signal Amplification involves using an enzyme to catalyze
a chemical reaction that amplifies the signal. In FIG. 5 a
non-conjugated primary antibody 100 bound to a cell of interest 102
is used, but instead of regular dye-conjugated 2.sup.nd antibody, a
2.sup.nd antibody 104 conjugated to an enzyme horse radish
peroxidase (HRP) 106 is used to target the primary antibody 100.
Then a reagent 108 containing a marker 110(*), such as a
fluorophore dye (e.g., Alexa 555), hydrogen peroxide H.sub.2O.sub.2
112(.DELTA.) and a tyramide mixture 114(.quadrature.) in a
non-reactive form is applied. HRP 106 in the presence of
(H.sub.2O.sub.2) 112 converts the non-reactive tyramide 114 into a
short-lived free-radical tyramide form 114'(.circle-solid.) that
deposits in the neighborhood of the site of the bound 2.sup.nd
antibody 104, through binding to nucleophilic protein tyrosine
sidechains. This free-radical mechanism allows multiple reactive
fluorophore-tyramides (110, 114') to be deposited, thus amplifying
the signal from dye 110. The short-lived nature of the converted
fluorophore-tyramides (110, 114') means they will not diffuse, and
will stay locally close to the 2.sup.nd types 104. In this scheme,
the dye 110 (e.g. Alexa 555) can be amplified many more times than
a dye (e.g., Alexa 488) directly conjugated to a second antibody
104.
The present procedure takes advantage of the amplification concepts
obtainable via use of TSA to create an asymmetric dye concentration
by incorporating the other dye (e.g., Alexa 488) 116 in the form of
a conjugate to other secondary antibody 104, at a level different
from dye 110, and in a particular embodiment, the amount of dye 116
would be less than the amount of dye 110. While TSA has been cited
as the amplification technique, other known biological
amplification techniques may also be used.
Turning to FIG. 6, shown is a further procedure to increase the
spectral resolution of a sample. In this example a first type
(primary) antibody 120 is, again, bound to a cell of interest 122
using known techniques. This procedure then uses a single
fluorophore dye 124 which is associated with a 2.sup.nd type
(secondary) of antibody 126, which itself is configured to target a
1.sup.st antibody 120. Single dye 124 is excited in a
non-conventional manner where the emission is collected and used in
a novel way.
In one example, dye 124 may be Alexa 555, and the Alexa 555
conjugated antibody 126 is excited by a 488 nm laser. Since its
excitation efficiency with a 488 nm laser is only 14.2% (see Table
1), slightly higher concentrations of Alexa 555 are used. For
example, instead of a regular 1000.times. dilution of the dye, a
150.times. dilution is used to compensate for the loss in
excitation efficiency. The dilution being the dilution of an
antibody-dye (or marker) in a buffer (such as phosphate buffered
saline) to be applied onto the sample under investigation. More
particularly, the dilution is in a range of six to seven times less
than the accepted industry standard of approximately 1000.times.
dilution. Then when the Alexa 555 is excited, sufficient
fluorescence around its regular emission peak (.about.585 nm) can
be effectively collected by the imaging system. An advantage of
this approach is the number of false positives stemming from blood
auto fluorescence (i.e., most of which occurs near an emission peak
around 525 nm when excited with a 488 nm laser) is dramatically
reduced. Since a moderate concentration is used, dye aggregates due
to high concentration of antibody are greatly reduced. Through
testing of concentration effects using cell lines of various
expression levels, false positives can be held to a manageable
level while all true positives are detected.
In an alternative procedure, fluorescence of these imaging events
may be undertaken and collected at a shorter emission wavelength
(e.g. 525 nm when using 488 nm laser) where most autofluorescence
occurs. Together with the emission at longer wavelength (e.g. 585
nm for Alexa 555 dye) their ratio provides an additional
distinctive set of data for filtering out false positives.
Another sample preparation procedure employs a tandem dye, such as
a commercially available tandem dye, DyeMer 488/630 (from Molecular
Probes Division of Invitrogen Corporation). For example, FIG. 7
illustrates the absorption curve 130 for the DyeMer 4881630 dye,
which shows it excites well at 488 nm besides its main absorption
peak around 615 nm; but has most of its emission centered around a
peak at 630 nm, as depicted by emission curve 150. Tandem dyes are
conjugates of two dyes, with the emission of the first dye being
absorbed by the second dye. Though a majority of the emission from
the first dye is absorbed, a small percentage can still be emitted
in its regular wavelength (i.e., 525 nm for Alexa 488). Therefore,
by collecting both emissions around 525 nm and 630 nm, a ratio
representing that of the tandem dye, rather than auto fluorescence
can be obtained. Coupled with other information such but not
limited to as features, size and/or intensity, and by applying a
set of filtering criteria, such as those previously discussed in
this document, a large number of the false positives from dye
aggregates are eliminated.
Using concepts as described above, during laser-based scanning
processes, the inventors have achieved a specificity of 10.sup.-5
and better. In other words, for a sample having approximately one
(1) million cells, false positives may be limited to just ten (10)
cells. These cells (true positives and reduced false positives
after filtering) may then be reviewed by a higher resolution
detection system such as a microscopic system in a short amount of
time.
It will be appreciated that various of the above-disclosed and
other features and functions, or alternatives thereof, may be
desirably combined into many other different systems or
applications. Also that various presently unforeseen or
unanticipated alternatives, modifications, variations or
improvements therein may be subsequently made by those skilled in
the art which are also intended to be encompassed by the following
claims.
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